Ultrasensitive Fluorescence Detection of DNA 

 in Sequencing Gels 



Richard A. Mathies and Konan Peck 



Chemistry Department, University of California, Berkeley, CA 94720 



(415)642-4192 



Our goal is to develop an ultrasensitive apparatus for the detection of fluorescently 

 labeled DNA fragments and probes in sequencing gels. A detection system has been 

 developed that is sensitive enough to observe the fluorescence emitted by individual 

 fluorescent molecules.' The idea is to detect the bursts of fluorescence arising from the 

 passage of individual molecules through a tightly focused laser beam. A theory for the 

 signal-to-noise ratio in laser-induced fluorescence has been developed and used to select 

 the optimal laser power and molecular transit time (Mathies et al., in preparation). This 

 theory has been satisfactorily tested by studying the fluorescence from phycoerythrin as 

 a function of the incident laser power and the transmit time of the molecule through the 

 laser beam. Using these optimized conditions, we have observed the fluorescence of 

 individual molecules of phycoerythrin in a flow system as they pass through the laser 

 beam. The calculated autocorrelation function of the detected photons clearly shows 

 that we are observing correlated bursts of fluorescence from individual molecules. Also, 

 the frequency of these bursts is linearly related to the concentration as expected for 

 single-molecule events. Using a hard-wired version of our single-molecule detector, we 

 have been able to detect phycoerythrin at concentrations as low as 1 x 10" M (Peck et 

 al., Proc. Natl. Acad. Sci. USA. in press). This detector is three orders of magnitude 

 more sensitive than conventional fluorescence detection systems. A patent on this on- 

 line, single-molecule counter has been filed.' We are now in the process of applying this 

 new technology to the detection of individual molecules on substrates and in gels. 



'R. A. Mathies, K. Peck and L. Stryer, "High Sensitivity Fluorescent Single Particle and 

 Single Molecule Detection Apparatus and Method," patent filed. 



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